NIH Mock Proposal
1. Specific Aims
2. Background and Significance
3. Research Design and Methods
   1. Yeast Genetics
   2. Immunopre- cipitation
   3. Yeast Two-Hybrid
   4. Future Experiments
4. References

3.2 Determination of other protein interactions with frataxin through immunoprecipitation

3.2.1 Co-immunoprecipitation

Immunoprecipitation experiments would allow the protein interactions to be confirmed. In order to carry out immunoprecipitation experiments, antibodies to YFH1 must be made. The yeast mutants overexpressing MFT1/2 have already been constructed with a Myc epitope at the C-terminus, so it is preferable to use another epitope tag for YFH1 such as influenza hemagglutinin (HA). Previously, the Myc epitope has been successfully added to the C-terminus of YFH1 with complete functionality of the protein, so it is expected that another epitope will work equally as well. The antibodies to HA will then be characterized to test the specificity to the YFH1-HA fusion protein. Using an epitope tag provides an easy method for protein identification by immunological methods; however, if there is a problem with specificity, antibodies to YFH1 and MFT1/2 can be made by injecting the protein into rabbits.

If the yeast experiments suggest that YFH1 and MFT1/2 interact, mitochondrial protein extracts of strains overexpressing both YFH1-HA and MFT1-myc or MFT2-myc can be immunoprecipitated with an anti-HA. Since protein A will bind to immunoglobins, beads conjugated to protein-A can be used to separate anti-HA and anything bound to it from the protein mixture. The immunoprecipitate can then be immunoblotted with anti-Myc to detect the presence of MFT1 or MFT2. If an interaction is detected, truncated forms of YFH1 can be used to detect which part of the protein interacts, and additional confirmation will be obtained by mutagenesis of residues in the regions involved in the interaction, thus disrupting the interaction.

If the yeast experiments suggest there is no direct interaction between YFH1 and MFT1/2, immunoprecipitation may allow for the identification of other possible proteins or can confirm the interaction of another protein. Yeast can be transformed with multicopy plasmids containing cDNA from yeast libraries. Then protein extracts can be immunoprecipitated with anti-HA as noted above. If this method is used, sequencing the corresponding plasmid DNA can identify the proteins pulled down. The interaction can then be verified as outlined above for the MFT1/2-YFH1 interaction. The possible disadvantage to this method is that some proteins are lethal when overexpressed.

If lethality is suspected to be problem, immunoprecipitation of mitochondrial protein extracts from cells expressing anti-HA may identify directly and indirectly interacting proteins. Proteins detected by this assay can be identified relatively easily since the S. cerevisiae genome sequence is readily available. To identify the interacting partners, protein bands can be digested in-gel with trypsin, and then the peptides can be sequenced by mass spectrometry.¹⁹ The sequences obtained can be matched to known sequence in the databases, and the full protein sequence can then be deduced.

3.2.2 Interpretation

The biggest pitfall of detecting an interaction by immunoprecipitation is that antibodies may react unspecifically to other proteins. Sometimes this can be difficult to avoid, but the HA and Myc epitopes are well characterized. Also, the stringency of the blotting procedure can be adjusted by altering the blocking times, the antibody concentration, and the wash solutions used. Confidence in the interaction is obtained after residues necessary for interaction have been identified.

It is expected that immunoprecipitation will yield results in detecting interacting proteins. Although it does take some time to develop and characterize antibodies, they are useful for later experiments once interacting proteins have been identified. Additionally, once antibodies are developed, the procedures involved in immunoprecipitation and immunoblotting are very straightforward and will allow research progress to continue in a timely fashion. As mentioned above, antibodies may recognize proteins in a non-specific manner. Adjusting the stringency of the reaction will help avoid problems of this sort. Additionally, the determination of the region necessary for interaction provides confidence in the results obtained.

3.2.3 Protocol

The YFH1-HA fusion construct will be made using basic PCR techniques. Protein extracts of yeast overexpressing YFH1-HA will be immunoblotted with the anti-HA; differing concentrations of anti-HA will be used to determine specificity for YFH1. Mitochondrial extracts from cells overexpressing MFT1/2 will be prepared as described previously¹⁸ for use in immunoprecipitation. Protein concentration can be normalized for the samples, and anti-YFH1 will be added. The dilution of anti-YFH1 will be determined based upon the specificity experiments. After incubation, the samples will be run on SDS-PAGE and then transferred to a PVDF membrane. The membrane is then blocked, washed, and incubated with anti-Myc. Then, after washing, a secondary antibody conjugated to horseradish peroxidase is added. The membrane can then be developed by a chemiluminescent solution and photographed. PCR-based site directed mutagenesis will help determine the actual residues involved in the interaction.

Antibodies will be made as described above. The procedures for immunoprecipitation are similar to that described above, except a mitochondrial extract will be prepared from wild type MFT1/2 strains of yeast. The use of mass spectrometry has been much simplified, and to accomplish protein identification in this manner, a collaborative effort is planned with the Faulkner Lab (SIO).

If necessary, Anti-YFH1 will be made using a protocol similar that used by Campuzano et al.⁷ for human frataxin antibodies. Briefly, YFH1 DNA can be amplified, adding restrictions sites as needed to the 5' and 3' ends to allow insertion into a prokaryotic expression vector such as pATH, which produces a TrpE-fusion protein. After inducing overexpression in E. coli, the protein construct can be isolated from inclusion bodies by solubilization in urea. The protein can be renatured by dialysis and injected into pathogen-free rabbits to produce antibodies, with booster injections as needed. The resulting antibodies will then be characterized.

Next Section: 3.3 Determination of protein interactions with frataxin through the yeast two-hybrid system

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